1858 • The Journal of Neuroscience, February 10, 2016 • 36(6):1858–1870 Neurobiology of Disease Altered Trafficking and Processing of GALC Mutants Correlates with Globoid Cell Leukodystrophy Severity Daesung Shin,1,2 M. Laura Feltri,1,2,3 and Lawrence Wrabetz1,2,3 1Hunter James Kelly Research Institute and Departments of 2Biochemistry and 3Neurology, Jacob School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14203 Globoid cell leukodystrophy (GLD, Krabbe disease) is due to autosomal recessive mutations in the lysosomal enzyme galactosylcerami- dase (GALC). Many GLD patients develop infantile-onset of progressive neurologic deterioration and death by 2 years of age, whereas others have a later-onset, milder disease. Cord blood transplant slows disease progression much more effectively when performed presymptomatically,highlightingtheimportanceofearlydiagnosis.CurrentdiagnosisisbasedonreducedGALCactivity,DNAsequence, and clinical examination. However, presymptomatic diagnosis is hampered by imperfect genotype-GALC activity-phenotype correla- tions. In addition, three polymorphisms in the GALC gene are variably associated with disease mutations and have unknown effects on GALC activity and disease outcome. Here, we study mutations that cause infantile or later-onset GLD, and show that GALC activity is significantly lower in infantile versus later-onset mutants when measured in the lysosomal fraction, but not in whole-cell lysates. In parallel, infantile-onset mutant GALCs showed reduced trafficking to lysosomes and processing than later-onset mutant GALCs. Finally, the cis-polymorphisms also affected trafficking to the lysosome and processing of GALC. These differences potentially explain why the activity of different mutations appears similar in whole-cell extracts from lymphocytes, and suggest that measure of GALC activity in lysosomes may better predict the onset and severity of disease for a given GLD genotype. Key words: galactosylceramidase; globoid cell leukodystrophy; Krabbe disease; lysosomal processing; lysosomal storage disorders; protein trafficking Significance Statement Globoid cell leukodystrophy (GLD, Krabbe disease) is diagnosed by measuring galactosylceramidase (GALC) activity and DNA analysis. However, genotype and phenotype often do not correlate due to considerable clinical variability, even for the same mutation, for unknown reasons. We find that altered trafficking to the lysosome and processing of GALC correlates with GLD severity and is modulated by cis-polymorphisms. Current diagnosis of GLD is based on GALC activity of total cell lysates from blood, which does not discriminate whether the activity comes from the lysosome or other subcellular organelles. Measurement of GALC activity in lysosomes may predict which infants are at high risk for the infantile phenotype while distinguishing other children who will develop later-onset phenotypes without onset of symptoms for years. Introduction lysosomal enzyme involved in the catabolism of galactosylcer- Globoid cell leukodystrophy (GLD, Krabbe disease) is a demyeli- amide, a major lipid in myelin, kidney, and epithelial cells of the nating, neurodegenerative disease that affects both the central small intestine and colon (Chen YQ et al., 1993). More than 85% and peripheral nervous systems, and is caused by mutations in of patients with GLD have the infantile form of the disease and the galactosylceramidase (GALC) gene. GALC (EC 3.2.1.46) is a present with symptoms between the ages of 3 and 12 months, which include extreme irritability, spasticity, developmental de- lay, and ultimately death within a few years. The remaining 10%– Received Aug. 17, 2015; revised Nov. 24, 2015; accepted Dec. 22, 2015. Author contributions: D.S., M.L.F., and L.W. designed research; D.S. performed research; D.S., M.L.F., and L.W. 15% of patients have a later-onset form of the disease, in which analyzed data; D.S., M.L.F., and L.W. wrote the paper. This work was supported by National Institutes of Health Grant R03-NS087359 to D.S., Western New York Stem Cell Pilot Studies to D.S., Empire State Development Corporation for Research Foundation-Krabbe Disease Research The authors declare no competing financial interests. WorkingCapitalGrantW753toL.W.andM.L.F.,EmpireStateDevelopmentCorporationforKrabbeDiseaseResearch CorrespondenceshouldbeaddressedtoDr.DaesungShin,HunterJamesKellyResearchInstitute,JacobSchoolof Capital Equipment Grant U446 to L.W. and M.L.F., and Hunter’s Hope Foundation. We thank Mirella Filocamo for Medicine and Biomedical Sciences, University at Buffalo, New York State Center of Excellence in Bioinformatics and information regarding Krabbe dermal fibroblast records; Christopher B. Eckman for providing the polyclonal GALC Life Sciences, 701 Ellicott Street, Buffalo, NY 14203. E-mail: [email protected]. antibody; Franca Cambi for providing the oli-neu cells; Edward Hurley for technical support; and Nadav I. Weinstock DOI:10.1523/JNEUROSCI.3095-15.2016 for useful comments on the manuscript. Copyright © 2016 the authors 0270-6474/16/361858-13$15.00/0 Shin et al. • Trafficking and Processing of Globoid Cell Leukodystrophy Mutants J. Neurosci., February 10, 2016 • 36(6):1858–1870 • 1859 symptoms present between 13 months of age and the seventh actgcggccg cgggttcggc-3Ј) and hGalc-cDNA-BamHI-as (5Ј-GGGGGG decade of life. Later-onset patients have a milder disease, which GGATCC CG gcgtgtggcttccacaagaaag-3Ј). GALC-HA was generated by may include weakness, loss of vision, and intellectual regres- modifying the hGalc-FLAG clone with the primers HA-fusion-SalI-s (5Ј- Ј sion (Wenger, 2011a). TCGACT ACCCAT ACGAC GTCCC AGACT ACGCT TAGG-3 ) and Ј Current therapy aims to reduce demyelination by hematopoi- HA-fusion-SalI-as (5 -TCGAC CTAAG CGTAG TCTGG GACGT CGT AT GGGTA G-3Ј). The QuikChange mutagenesis kit (Stratagene) was used etic cell transplantation (HCT), which generates cells that are for site-directed mutagenesis, and the primers used were hGalc-change of thought to transfer GALC to myelinating cells. However, judging glycine to aspartate at the amino acid residue number 286 (G286D)-s (5Ј- the efficacy of cord blood transplantation is complicated by gacatgggtgcagactgctggggtcgc-3Ј), hGalc-G286D-as (5Ј-gcgaccccagcagtctg- variable genotype–phenotype relationships in these patients. cacccatgtc-3Ј), hGalc-change of threonine to methionine at the amino acid Furthermore, for effective treatment in children with the residue number 529 (T529M)-s (5Ј-cctggcgagcatcacttcatgctacgccaag-3Ј), infantile-onset phenotype, transplantation must be performed hGalc-T529M-as (5Ј-cttggcgtagcatgaagtgatgctcgccagg-3Ј), hGalc-change of before the onset of symptoms (Duffner et al., 2012), but at this tyrosine to serine at the amino acid residue number 567 (Y567S)-s (5Ј- age HCT is associated with a 20% mortality rate. Therefore, ac- tgactacaaagtgtgatgttagcatagagacccctgacacag-3Ј), hGalc-Y567S-as (5Ј- curate diagnosis and prognosis are extremely important for the ctgtgtcaggggtctctatgctaacatcacactttgtagtca-3Ј), hGALC-change of glycine to serine at the amino acid residue number 57 (G57S)-s (5Ј-ccgcgccgatgctgtc- care of GLD patients. Ј Ј Ј Unfortunately, neither measurements of GALC enzymatic ac- gaactcccg-3 ), hGALC-G57S-as (5 -cgggagttcgacagcatcggcgcgg-3 ), hGALC-change of aspartate to asparagine at the amino acid residue number tivity nor documentation of GALC mutations reliably predicts 544 (D544N)-s (5Ј-gattgtgttggatgcattggcagcccatgtaatgg-3Ј), hGALC- the severity of phenotype in GLD patients. For example, in new- D544N-as (5Ј-ccattacatgggctgccaatgcatccaacacaatc-3Ј), hGALC-change of born screening, an infant with a low GALC activity and two doc- leucine to serine at the amino acid residue number 634 (L634S)-s (5Ј- umented mutant alleles may be diagnosed with the infantile form aggtgaaatgacccttaatagttgacgtgagtgtataccattttttt-3Ј), and hGALC-L634S-as of GLD, when indeed, the child may have the later-onset form of (5Ј-aaaaaaatggtatacactcacgtcaactattaagggtcatttcacct-3Ј) for the mutations. the disease. This child would accordingly undergo HCT and in- For the polymorphisms, hGalc-C550T-s (5Ј-ggattgtgggcgccaagtgttaccat- creased risk of mortality, despite the fact that disease would have gatttggac-3Ј), hGalc-C550T-as (5Ј-gtccaaatcatggtaacacttggcgcccacaatcc-3Ј), appeared significantly later. hGalc-G742A-s (5Ј-cgaactcttcaaggtggttaatgttataggggctcatt-3Ј), hGalc- Ј Ј The goal of this study is to understand why mutations with a G742A-as (5 -aatgagcccctataacattaaccaccttgaagagttcg-3 ), hGalc-C1685T-s Ј Ј range of predicted severity do not always correlate with differ- (5 -tacaactggaccaatctgactataaagtgtgatgtatacatagag-3 ), and hGalc- C1685T-as (5Ј-ctctatgtatacatcacactttatagtcagattggtccagttgta-3Ј) were used. ences in GALC activity, and why the same mutation in GALC can All the constructs were confirmed by DNA sequencing. result in a broad spectrum of clinical variability. Three factors Animal cell culture and transfection. All animal experimentation was have been suggested as potential contributors to clinical variabil- performed in strict accord with Institutional Animal Care and Use Com- ity: cis-polymorphisms (Raghavan et al., 2005), environmental mittee approved protocols (Roswell Park Cancer Institute and University influences including diet (Pannuzzo et al., 2010), and modifier at Buffalo). HEK-293T cells were maintained in DMEM supplemented genes such as saposinA/Psap (Yagi et al., 2004) or others (Lattanzi with 10% FBS. Primary oligodendrocyte progenitor cell